The objectives are to define the structural organization of the centromere regions of eukaryotic chromosomes, and to understand the molecular mechanisms involved in chromosome segregation during mitotic and meiotic cell divisions. The centromere studies supported by this grant are carried out using as experimental systems the budding yeast, Saccharomyces cerevisiae, and the filamentous fungus, Aspergillus nidulans. Synthetic minichromosomes, containing a functional centromere (CEN), a DNA replicator function, and telomeric DNA (if linear), constitute invaluable probes for the study of chromosome structure and function. Specific aims include: (1) the construction of structurally altered centromere DNAs with altered function, using in vitro mutagenesis techniques; (2) the identification of genes specifying kinetochore structural proteins or gene products necessary for formation of the yeast kinetochore; (3) the isolation and characterization of kinetochore proteins (centromere binding proteins); (4) investigation of the mechanism of kinetochore-microtubule associations; (5) investigation of specialized DNA structures in the centromere region, including replicative intermediates, kinked DNA, and methylated DNA; (6) isolation and structural characterization of centromere DNAs from Aspergillus nidulans, as a step toward an understanding of centromere structure-function in more complex organisms. Studies on the molecular mechanisms involved in cell division and chromosome segregation in the experimentally tractable lower eukaryotes offer an approach to an understanding of analogous mechanisms as they function normally and abnormally in higher organisms.